Dipeptidyl peptidase I (DPPI, EC 3.4.14.1), previously known as dipeptidyl aminopeptidase I (DAPI), dipeptidyl transferase, cathepsin C and cathepsin J is a lysosomal cysteine exo-peptidase belonging to the papain family. DPPI is widely distributed in mammalian and bird tissues and the main sources of purification of the enzyme are liver and spleen. The cDNAs encoding rat, human, murine, bovine, dog and two Schistosome DPPIs have been cloned and sequenced and show that the enzyme is highly conserved. The human and rat DPPI cDNAs encode precursors (preproDPPI) comprising signal peptides of 24 residues, proregions of 205 (rat DPPI) or 206 (human DPPI) residues and catalytic domains of 233 residues which contain the catalytic residues and are 30-40% identical to the mature amino acid sequences of papain and a number of other cathepsins including cathepsins L, S, K, B and H.
The translated preproDPPI is processed into the mature form by at least four cleavages of the polypeptide chain. The signal peptide is removed during translocation or secretion of the proenzyme (proDPPI) and a large N-terminal proregion fragment, which is retained in the mature enzyme, is separated from the catalytic domain by excision of a minor C-terminal part of the proregion, called the activation peptide. A heavy chain of about 164 residues and a light chain of about 69 residues are generated by cleavage of the catalytic domain.
Unlike the other members of the papain family, mature DPPI consists of four subunits, each composed of the N-terminal proregion fragment, the heavy chain and the light chain. Both the proregion fragment and the heavy chain are glycosylated.
DPPI catalyses excision of dipeptides from the N-terminus of protein and peptide substrates, except if (i) the amino group of the N-terminus is blocked, (ii) the site of cleavage is on either side of a proline residue, (iii) the N-terminal residue is lysine or arginine, or (iv) the structure of the peptide or protein prevents further digestion from the N-terminus.
DPPI is expressed in many tissues and has generally been associated with protein degradation in the lysosomes. More recently, DPPI has also been assigned an important role in the activation of many granule-associated serine proteinases, including cathepsin G and elastase from neutrophils, granzyme A, B and K from cytotoxic lymphocytes (CTL, NK and LAK cells) and chymase and tryptase from mast cells. These immune/inflammatory cell proteinases are translated as inactive zymogens and the final step in the conversion to their active forms is a DPPI-catalysed removal of an activation dipeptide from the N-terminus of the zymogens. DPPI -/- knock-out mice have been shown to exclusively accumulate the inactive, dipeptide extended proforms of the pro-apoptopic proteases granzyme A and B.
Many of the granule-associated proteases, which are activated by DPPI, serve important biological functions and inhibition of DPPI may thus be a general means of controlling the activities of these proteases.
Neutrophils cause considerable damage in a number of pathological conditions. When activated, neutrophils secrete destructive granular enzymes, including elastase and cathepsin G, and undergo oxidative bursts to release reactive oxygen intermediates. Numerous studies have been conducted on each of these activating agents in isolation. Pulmonary emphysema, cystic fibrosis and rheumatoid arthritis are just some examples of pathological conditions associated with the potent enzymes elastase and cathepsin G. Specifically, the imbalance in plasma levels of these two enzymes and their naturally occurring inhibitors, alpha 1-protease inhibitor and antichymotrypsin, may lead to severe and permanent tissue damage. These facts together with the shown relation between the induction of neutrophil activation and the activation and release of elastase and cathepsin G point to DPPI as an alternative target enzyme for therapeutic intervention against rheumatoid arthritis and related autoimmune diseases.
Cytotoxic lymphocytes play an important role in host-cell responses against viral and intracellular bacterial pathogens. They are also involved in anti-tumour responses, allograft rejection, and in a number of various autoimmune diseases. Though CTL, NK, and LAK cells kill via multiple mechanisms, evidence over the past few years have shown that two major pathways are responsible for the induction of target cell apoptosis. These are the Fax-FasL pathway and the granule exocytosis pathway.
Activated cytotoxic lymphocytes contain lytic granules, which are the hallmark of specialised killer cells. Among the proteins found in lytic granules are perforin and the highly related serine proteases of the granzyme family, including granzyme A, B and K. The importance of perforin and granzymes for cell-mediated cytotoxicity and apoptosis has been firmly established in several loss-of-function models.
Granzyme A and B knockout mice have shown that granzyme B is critical for the rapid induction of apoptosis in susceptible target cells, while granzyme A plays an important role in the late pathway of cytotoxicity. The above mentioned fact that DPPI -/- knock-out mice have been shown to exclusively accumulate the inactive proforms of granzyme A and B points to DPPI as an alternative target enzyme for therapeutic intervention and also provides a rationale for developing inhibitors against DPPI that could modulate immune responses against tumours, grafts, and various autoimmune diseases.
Mast cells are found in many tissues, but are present in greater numbers along the epithelial linings of the body, such as the skin, respiratory tract and gastrointestinal tract. Mast cells are also located in the perivascular tissue surrounding small blood vessels. This cell type can release a range of potent inflammatory mediators including cytokines, leukotrienes, prostaglandins, histamine and proteoglycans. Among the most abundant products of mast cell activation, though, are the serine proteases of the chymotrypsin family, tryptase and chymase. The use of in vivo models has provided confirmatory evidence that tryptases and chymases are important mediators of a number of mast cell mediated allergic, immunological and inflammatory diseases, including asthma, psoriasis, inflammatory bowel disease and atherosclerosis. For years, pharmaceutical companies have targeted the inhibition of tryptase and chymase as a drug intervention strategy.
However, the active sites and catalytic activities of tryptases and chymases closely resemble a number of other proteases of the same family and it has proven very difficult to design inhibitors that are at the same time sufficiently selective, potent, non-toxic and bioavailable. Furthermore, the large quantities of tryptases and chymases that are synthesised and released by mast cells make it difficult to ensure a continuous and satisfactory supply of inhibitors at the sites of release. The strong evidence associating tryptases and chymases with a number of mast cell mediated allergic, immunological and inflammatory diseases, and the fact that DPPI is needed for the activation of tryptase and chymase, outline DPPI as an alternative target enzyme for therapeutic intervention against the above mentioned mast cell diseases.
Low molecular weight substrates that mimic peptidyl inhibitors of DPPI, such as Gly-Phe- and Gly-Arg- diazomethyl ketones, chloromethyl ketones and fluoromethyl ketones have previously been reported. However, due to their peptidic nature and reactive groups, such inhibitors are typically characterised by undesirable pharmacological properties, such as poor oral absorption, poor stability, rapid metabolism and high toxicity.
Knowledge of the crystal structure co-ordinates and atomic details of DPPI, or its mutants or homologues or co-complexes, would facilitate or enable the design, computational evaluation, synthesis and use of DPPI inhibitors with improved properties as compared to the known peptidic DPPI inhibitors.
In addition to the interest in the unique structural and functional properties of DPPI, attention has also been turned to the technological applications of the enzyme.
By virtue of its restricted specificity, DPPI has been shown to be suitable for excision of certain extension peptides from the N-termini of recombinant proteins having a DPPI stop-point integrated in or placed in front of their N-terminal sequences. These properties of DPPI have been utilised to develop a specific and efficient method using recombinant DPPI variants for complete removal of a group of purification tags from the N-termini of target proteins. The addition of purification tags to the target protein is a simple and well-established approach for generating a novel affinity, making one-step purifications of recombinant proteins possible by using affinity chromatography. The combined processes of using purification tags for purification of recombinant proteins and DPPI for cleavage of the purification tag generating the desired N-terminal in the target protein (the DPPI/tag strategy), hold promises for use in large-scale productions of pharmaceutical proteins and peptide products. Its strength obviously is the simple overall design, the use of robust and inexpensive matrices, and the use of efficient enzymes.
In order to fully exploit the potential of this DPPI/tag strategy, it is thus desirable to alter the chemical, physical and enzymatic properties of DPPI to be able to use the enzyme in different condition, thereby making the DPPI/tag strategy more efficient, flexible and/or even more economically feasible.
Furthermore, besides its aminopeptidase activity, DPPI also displays a transferase activity, i.e. DPPI catalyses the transfer of dipeptide moieties from amides and esters of dipeptides to the N-terminal of unprotected peptides and proteins. This transferase activity of DPPI consequentely bears a potential usage in methods for enzymatic synthesis and/or semisynthesis of peptides and proteins, but because of problems with the reverse (aminopeptidase) activity and substrate restrictions, transpeptidation by DPPI has been rarely used or exploited for peptide and protein synthesis.
The crystal structure of a number of cysteine peptidases of the papain family, including papain, chymopapain, actinidin, cathepsin B, and cathepsin have been known for many years, but despite DPPI being highly homologous to the other members of the papain family, and despite DPPI being available as purified and characterised preparation since 1960 (Metrione, R. M. et al, Biochemistry 5,1597-1604, 1966; McDonnald J. K. et al, J. Biol. Chem. 244, 2693-2709, 1969), it has until now been impossible to obtain crystals of DPPI for solving the crystal structure of the enzyme.
Alternative interests have thus been focussed on trying to solve some of the structural features of DPPI through homology modelling, based on the known crystal structures of other cysteine peptidases of the papain family. However, although there are many resemblances to these other cysteine peptidases, it has not been possible to model the structure of DPPI because of very distinct differences. These differences include the oligomeric structure of DPPI, the detainment of the residual propart in the active enzyme and a unique chain cleavage pattern in active DPPI, features not present in and/or seen in the known crystal structures of the other cysteine peptidases of the papain family.